Gel Chromatography, also known as Size Exclusion Chromatography (SEC), is a technique that separates molecules based on size.
It is widely used in the pharmaceutical industry for analyzing and purifying biomolecules such as proteins, peptides, nucleic acids, and polysaccharides.
Column Composition: Packed with porous beads made of materials like agarose, dextran, or polyacrylamide.
Separation Mechanism: Smaller molecules enter the bead pores, eluting slower; larger molecules bypass the pores and elute faster.
Principle
Gel Chromatography operates on the size exclusion principle, where separation depends on a molecule's size or hydrodynamic volume:
Large Molecules: Cannot enter the pores, elute quickly.
Intermediate Molecules: Partially enter pores, elute at intermediate times.
Small Molecules: Enter most or all pores, elute last.
Separation is based on molecular size in solution, not intrinsic size.
Classification of Gel Chromatography
Based on Material and Use:
Gel Filtration Chromatography (GFC): For aqueous samples; uses hydrophilic gels (e.g., dextran, agarose); applied in protein purification.
Gel Permeation Chromatography (GPC): For synthetic polymers in organic solvents; uses hydrophobic beads (e.g., polystyrene).
Based on Pore Size:
Micro-Porous: For low molecular weight compounds.
Meso-Porous: For medium molecular weight compounds.
Macro-Porous: For high molecular weight compounds.
Based on Usage:
Analytical Chromatography: Analyzes molecular weight, size, and purity.
Preparative Chromatography: Purifies and collects specific molecules in large quantities.
Advantages:
Mild Conditions: Works under non-denaturing conditions, preserving the biological activity of sensitive molecules like enzymes or proteins.
Versatility: Can be used for a wide range of molecules from small ions to large proteins or nucleic acids.
High Resolution: Can effectively separate molecules with minor size differences.
Desalting Capability: Efficient in removing small molecules like salts from larger ones in a sample.
Disadvantages:
Limited Range: Each column has a specific molecular weight range. Molecules outside this range might not be separated efficiently.
Bead Degradation: Over time, or under harsh conditions, the beads can degrade or shrink.
Limited Chemical Compatibility: Some gels can be incompatible with certain solvents or conditions.
Sample Recovery: Sometimes, the sample might interact non-specifically with the matrix, leading to reduced recovery.